Activation of resting T lymphocytes through the T cell antigen receptor (TCR) is initiated by rapid, but transient, phosphorylation of several regulatory proteins or enzymes on tyrosine residues. T cell activation is greatly aided by co-stimulation of the CD28 molecule, which also uses one or several protein tyrosine kinases (PTKs). At least six PTKs belonging to the Src, Syk, Csk and Tec families are known to be involved in the earliest events induced by these receptors. Only one protein tyrosine phosphatase (PTPase), CD45, has so far been found to be important, while two other PTPases, SHP1 and SHP2, may participate. There is some evidence that SHP1 acts as a negative regulator of TCR signaling, while SHP2 may play a positive role by acting as a bridge to the Grb2-Sos-Ras pathway. This proposal focuses on SHP1 and SHP2. Specific Aim 1 attempts to elucidate the role, importance and regulation of SHP1 and SHP2 in the initial/early events of TCR/CD3 plus CD28- induced T cell activation. First, we will use a number of rapid transfection / reporter assays. Preliminary findings indicate that both SHP1 and SHP2 will yield positive results in these assays. In-depth investigations of these findings will also include stably transfected cell lines overexpressing wild-type, catalytically inactive or substrate trapping mutants of SHP1 and SHP2. We will further address the mechanisms by which these molecules participate in signal transduction, including the determination of subcellular location, possible receptor associations, protein-protein interactions, identification of tyrosine phosphorylation sites and measurement of catalytic activities. The biological relevance of our findings will be evaluated using site- directed mutagenesis. Specific Aim 2 examines the effects of SHP1 and 2 on specific intracellular targets upon TCR and/or CD28 stimulation. Experiments will identify the physiological targets in a systematic manner. Some of these are already suggested by preliminary data. Further experiments will study these findings in detail, identify the sites of binding and dephosphorylation and effects of dephosphorylation. We will also investigate the finding that SHP2 upon tyrosine phosphorylation, binds the Grb2 adapter protein, which, in turn, couples to the Sos/Ras pathway. In addition, Grb2 brings a number of potential substrates into proximity of SHP2. This potential mechanism of regulated access to substrates will be investigated. We anticipate that the results obtained in this study will enable us to better understand the molecular events that follow TCR plus CD28 triggering and govern the onset of T cell proliferation.